1. Field of the Invention
The present invention generally relates to fluorescence measurements. More specifically, the present invention relates to frequency domain measurements of the fluorescence lifetime and the fluorescence spectrum.
2. Description of the Related Art
The process of fluorescence occurs when a substance, such as a molecule, absorbs light at one wavelength (or energy), and then emits light at a longer wavelength (or lower energy). A slight time delay occurs from when the substance absorbs light and when the substance re-emits light at the longer wavelength. This time delay is known as the fluorescence lifetime.
FIG. 1 depicts the fluorescence process schematically for a diatomic molecule, which is a molecule that is comprised of two atoms. There are several "types" of energy that are internal to a diatomic molecule, one of which is vibrational energy. The classic example of vibrational energy is imagining the two atoms connected together by a spring where the atoms oscillate back and forth along the axis of the spring. As the two atoms approach each other, they experience repulsion due to the proximity of their negatively charged electron clouds. As the atoms pull apart from one another, they experience an attraction that one can imagine as the result of the attractive forces between the positively charged nucleus of one atom for the negatively charged electron cloud of the other atom, and vice versa. The distance between the two atoms is known as the internuclear distance, which constantly changes as the two atoms oscillate back and forth. As the internuclear distance decreases, a rise in potential energy occurs due to the repulsive forces; as the internuclear distance increases, a rise in potential energy again occurs due to the attractive forces. FIG. 1 illustrates this difference in potential energy by line 11 in the potential energy "well" 10 and by line 13 in the potential energy "well" 12 where line 11 and 13 plot the potential energy as a function of internuclear distance.
A group of horizontal lines 20, 22, and 24 appear in each of the potential energy wells 10 and 12. Each horizontal line represents the individual vibrational energy states possible for the diatomic molecule. Quantum mechanics requires that the vibrational frequency of the spring be within certain values. In other words, the spring may oscillate at frequency `a`, and the spring may oscillate at frequency `b`, but it is a physical impossibility for the spring to oscillate at any frequency between `a` and `b`. In FIG. 1, any two adjacent horizontal lines would represent `a` and `b`.
The different potential energy wells 10 and 12 represent another type of energy in the diatomic molecule: electronic energy. Different electronic energies occur when the molecule absorbs energy in such a fashion that it causes an electron to move to a higher energy configuration within the molecule. The classical example of the energy absorption is the `changing the spring` that connects the two atoms. If we "add" electronic energy to the potential energy well 10, the energy raises the potential energy well 10 to the energy level of potential energy well 12.
With this background, we can use FIG. 1 to describe the process of fluorescence and fluorescence lifetime for a diatomic molecule. The example molecule is originally in a state depicted by the lower potential energy well 10. The molecule absorbs a photon of the correct amount of energy that induces an electron to move to a higher electronic energy state, represented by the upper potential energy well 12. The diatomic molecule also tends to undergo a change from a `lower vibrational state` (the horizontal lines 20 and 22) in the lower potential well 10 to a higher vibrational state in the upper potential well 12. This initial excitation of the molecule is shown by line 14. Through any one of numerous possible processes, the higher vibrational state 24 in the upper well decays to a lower vibrational state 22 in the upper well (as shown by line 18). The process of non-radiative decay to a lower vibrational energy level in the upper well occurs very rapidly. After the non-radiative vibrational relaxation, the electron will want to return or revert back to a lower energy state as shown by line 16 (in other words, the electron wants to move back to where it was before the whole process started), and in so doing, the molecule emits a photon of light (with a lesser amount of energy than the excitation photon). This lowering of the energy state and the emission of a photon is the process of fluorescence. The process of emitting the photon has an associated time delay, which is the fluorescence lifetime.
In practice, we typically probe many different types of molecules at once with the excitation light pulse. FIG. 2 illustrates the case where a single short excitation pulse of light 30 is absorbed by a sample of identical molecules all at once. The fluorescence decay curve 32 resulting from a typical fluorescence response of a sample of identical molecules is exponential in nature because not all of the identical molecules emits its fluorescence photon at precisely the same time. The exponential decay follows a mathematical function so that we can calculate the fluorescence lifetime, .tau.: EQU I(t)=I.sub.0 .multidot.e.sup.-(t-5.sbsp.0.sup.)/.tau.
where t.sub.0 is the time the excitation pulse, I.sub.0 is the initial fluorescence and I(t) is the observed fluorescence intensity as a function of time.
In the cases with multiple molecular types (where more than one lifetime decay is present within a sample of a target mixture), the exponential decay seen in FIG. 2 would appear as a sum of exponential functions. Prior art fluorometers typically use a type of time-correlated (or time-resolved) system that count the emitted photons (from an excited molecule) in order to measure the fluorescence lifetime. Other prior art systems add the ability to take the fluorescence lifetime measurements in the frequency domain by modulating the dynode of a photomultiplier tube, followed by a mixing and correlation procedure. These prior art systems are cumbersome, time consuming, and complicated to operate. The present invention overcomes the limitations of the prior art systems by utilizing a novel technique to measure the fluorescence lifetime and spectrum. Instead of irradiating the target sample with a single short pulse of light (photon counting), the present invention continuously irradiates the target sample with a light source whose amplitude modulation frequency is stepped with time. This technique allows us to use a chemometric analysis to automatically extract the lifetimes from the `phase delay` and `intensity vs. frequency` characteristics of the emitted light.